The occurrence of Brachyspira species and Lawsonia intracellularis was investigated by PCR analyses of faeces from 60 children living on European pig farms. In addition, 60 other children were included as controls. Two samples were positive for B. aalborgi but B. pilosicoli and L. intracellularis were not demonstrate

BENZ, M.

BRAUN-FAHRLÄNDER, C.

FELLSTRÖM, C.

FLÖISTRUP, H.

JACOBSON, M.

RIEDLER, J.

RÅSBÄCK, T.

SCHRAM-BIJKERK, D.

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SHORT REPORTSurvey on the occurrence of Brachyspira species and Lawsoniaintracellularis in children living on pig farmsM. JACOBSON 1*, T. RA˚SBA¨CK1, H. FLO¨ISTRUP2, M. BENZ 3,C. BRAUN-FAHRLA¨NDER 4, J. RIEDLER 5, D. SCHRAM-BIJKERK 6AND C. FELLSTRO¨M 11 Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Uppsala, Sweden2 Division of Environmental Epidemiology, Institute of Environmental Medicine, Karolinska Institute,Stockholm, Sweden3 Dr. von Haunersches Kinderspital, University Children’s Hospital, Mu¨nchen, Germany4 Department of Environment and Health Institute of Social and Preventive Medicine, Universityof Basel, Switzerland5 Children’s Hospital Schwarzach, Schwarzach, Salzburg, Austria6 Institute for Risk Assessment Sciences, University of Utrecht, The Netherlands(Accepted 4 October 2006; ﬁrst published online 30 November 2006)SUMMARYThe occurrence of Brachyspira species and Lawsonia intracellularis was investigated by PCRanalyses of faeces from 60 children living on European pig farms. In addition, 60 other childrenwere included as controls. Two samples were positive for B. aalborgi but B. pilosicoli andL. intracellularis were not demonstrated.Two species of the genus Brachyspira, B. aalborgi andB. pilosicoli, have been associated with human intes-tinal spirochaetosis (HIS) [1, 2]. The clinical signiﬁ-cance of HIS is largely unknown. In Westerncountries, B. aalborgi seems to be more common withreported prevalences of for example, 7.9% [3] or 70%of submitted cases of HIS [4]. Brachyspira pilosicoli ismore frequent in developing countries with reportedprevalences of 15–23% [2, 3, 5]. Further, B. pilosicoliand the intracellular rod Lawsonia intracellularis aremajor causes of enteric disease in young, growing pigswith 32% and 48%, respectively, of the herds beinginfected [6, 7]. The infection causes similar clinicalsigns and the microbes often occur concomitantly.L. intracellularis have been isolated in a wide range ofother animal species, e.g. pig, hamster, horse, guineapig, dog, lamb, calf, ferret, fox, deer, rabbit, rat,mouse, ratites, wild boar, wolf, giraﬀe, hedgehog, andprimates. The broad host range raises the questionwhether natural cross-species transmission mightoccur. L. intracellularis belongs to the Desulfovi-brionaceae family and in patients with ulcerative col-itis an increased carriage of closely related bacteriahas been demonstrated. The microbe causes prolifer-ative enteropathy, characterized by crypt hyperplasiaof immature cells. The lesions are morphologicallysimilar to those found in patients with coeliac diseaseand hypotheses regarding a similar aetiology havebeen proposed. However, the bacterium has neverbeen reported in humans [8–10].The aim of the present study was to investigatethe occurrence of Brachyspira species and L. intra-cellularis in faeces from children living on Europeanpig farms and compare the ﬁndings to those inchildren not living on pig farms.* Author for correspondence: Dr M. Jacobson, Department ofClinical Sciences, Faculty of Veterinary Medicine and AnimalScience, PO Box 7018, SE-750 07 Uppsala, Sweden.(Email : Magdalena.Jacobson@kv.slu.se)Epidemiol. Infect. (2007), 135, 1043–1045. f 2006 Cambridge University Pressdoi:10.1017/S0950268806007606 Printed in the United Kingdomhttps:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268806007606Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 20:02:44, subject to the Cambridge Core terms of use, available atThe study was conducted as part of a cross-sectional study on factors characteristic of anthro-posophic and farming populations, focusing onchildren from these groups. The study was approvedby the Regional Ethical Review Board, Stockholm,Sweden. Five European countries participated and 60of the children included in the study were living on pigfarms. Seven children were from Austria, 14 fromGermany, 14 from The Netherlands, nine fromSweden and 16 from Switzerland. The mean age was 9years (range 6–14 years). On average, each farm kept12 pigs. All children had been in contact with the pigs.Moreover, 60 children of similar age and from thesame geographic areas, but not living on pig farms,were included as controls in the present study. Ofthese, 30 were living on farms keeping other livestockthan pigs, and 30 children were living in the samegeographic areas but not on farms. Stool sampleswere collected in insulated bags, transported on iceand stored in a refrigerator (x80 xC) until requiredfor processing. DNA was prepared from faeces by theuse of a commercial kit (QIAamp1 DNA Stool Minikit ; Qiagen Inc., Valencia, CA, USA). The detectionof Brachyspira spp. were performed as described byKraatz et al. [4]. Genus-speciﬁc primers targeting 16SrDNA were used in PCR and species determinationwas performed by sequencing of the PCR amplicons.Demonstration of L. intracellularis was carried out byPCR using speciﬁc primers directed to chromosomalDNA according to Jones et al. [11]. An internalcontrol (mimic) was included in each tube to detectPCR inhibition. The sensitivity to detect the mimic infaecal sample preparations was 102 mimics per PCR[11–13].Two samples, one from a 12-year-old boy living ona pig farm and one from a 9-year-old girl not living ona farm, both from Switzerland, were found positivefor B. aalborgi by PCR and sequencing. In both chil-dren, strains similar to the type strain, strain W1 anda clone designated Hcc33 (GenBank accession num-bers Z22781, AF200693, and AF228813, respectively)were identiﬁed. None of the children had been absentfrom school because of illness or treated with anti-biotics during the last 3 months prior to sampling.Neither Brachyspira pilosicoli or L. intracellularis wasdetected in any sample. In one sample from a childliving on a pig farm and in three samples fromchildren in the control group the PCR was inhibited,as judged by the absence of the mimic amplicon.The mimic was visualized in the negative controlsincluded.B. aalborgi is generally the most commonBrachyspira species found in Western societies [14].This is in accordance with the present study, whereonly B. aalborgi was isolated. The clinical signiﬁcanceof the infection in humans has not been clariﬁed[3, 15]. In the present study, no illness was recordedin the infected children. Further, it was not possibleto relate the infection to husbandry. Isolates ofB. aalborgi has been demonstrated in humans, non-human primates and opossums [16] and the ability tocolonize might be determined by indigenous factorssuch as body temperature. Although commonlyfound in several animal species, it has not been poss-ible to relate ﬁndings of B. pilosicoli to the keeping ofpigs [2]. Human isolates have, however, previouslybeen shown to cause disease in pigs and chickens inexperimental challenge studies and cross-speciestransmission may occur naturally [17, 18]. In thepresent study, no samplings were performed in thepig herds and the occurrence of these bacteria inthe herds is therefore unknown. Hence, it is notpossible to draw any conclusions regarding the riskfor children living on pig farms to contract thesediseases.The microbe L. intracellularis have a very broadhost range, including non-human primates, and theinfection does not seem to be related to the host’sbody temperature. Experimentally, cross-speciestransmission has been demonstrated in pig, horse,hamster and mice [19]. Hence, it does not seem un-likely that this microbe would also be capable of in-fecting humans. However, in the present study thebacterium was not detected. It is possible that chil-dren living under poor hygienic circumstances, inclose contact with highly infected faeces, would havebeen a more suitable target in this respect.ACKNOWLEDGEMENTSThe work was supported by grants from theEuropean Union QLRT 1999-01391 and by fundingfrom the Swedish Foundation for Health CareScience and Allergy Research. Thanks are due to DrLars Engstrand at the Department of InfectiousDiseases, Solna, for allowing us the use of their fa-cilities.DECLARATION OF INTERESTNone.1044 M. Jacobson and othershttps:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268806007606Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 20:02:44, subject to the Cambridge Core terms of use, available atREFERENCES1. Hovind-Hougen K, et al. Intestinal spirochaetosis :morphological characterization and cultivation of thespirochete Brachyspira aalborgi gen. nov., sp. nov.Journal of Clinical Microbiology 1982; 16 : 1127–1136.2. Trott DJ, et al. The prevalence of Serpulina pilosicoli inhumans and domestic animals in the Eastern Highlandsof Papua New Guinea. Epidemiology and Infection1997; 119 : 369–379.3. Brooke CJ, Riley TV, Hampson DJ. The prevalence ofintestinal spirochaetes in Western Australians. InProceedings of the Second International Conference onColonic Spirochaetal Infections in Animals and Humans.Eddlestone, Scotland, UK, 2003, p. 7.4. Kraatz W, et al. Human intestinal spirochetosis diag-nosed with colonoscopy and analysis of partial 16SrDNA sequences of involved spirochetes. AnimalHealth Research Reviews 2001; 2 : 111–116.5. Margawani KR, et al. Prevalence, risk factors and mol-ecular epidemiology of Brachyspira pilosicoli in humanson the island of Bali, Indonesia. Journal of MedicalMicrobiology 2004; 53 : 325–332.6. Jacobson M, et al. Diarrhoea in the growing pig – acomparison of clinical, morphological and microbialﬁndings between animals from good and poor per-formance herds. Research in Veterinary Science 2003;74 : 163–169.7. Jacobson M, et al. The prevalence of Brachyspira spp.and Lawsonia intracellularis in Swedish swine herds andin the wild boar population. Journal of VeterinaryMedicine B 2005; 52 : 386–391.8. Cooper DM and Gebhart CJ. Comparative aspects ofproliferative enteritis. Journal of American VeterinaryMedical Association 1998; 212 : 1446–1451.9. Pitcher MCL, et al. Ulcerative colitis and porcine pro-liferative enteropathy: a common bacterial etiology?Proceedings of the Annual Meeting of the AmericanGastroenterology Association 1995; 108 : A894.10. Smith DGE, et al. Gamma interferon inﬂuences intesti-nal epithelial hyperplasia caused by Lawsonia intra-cellularis in mice. Infection and Immunity 2000; 68 :6737–6743.11. Jones GF, et al. Enhanced detection of intracellu-lar organism of swine proliferative enteritis, ilealsymbiont intracellularis, in feces by polymerase chainreaction. Journal of Clinical Microbiology 1993; 31 :2611–2615.12. Jacobson M, et al. Routine diagnostics of Lawsoniaintracellularis performed by PCR, serological and postmortem examination, with special emphasis on samplepreparation methods for PCR. Veterinary Microbiology2004; 102 : 189–201.13. Jacobson M, Englund S, Ballagi-Porda´ny A. The use ofa mimic to detect polymerase chain reaction-inhibitoryfactors in feces examined for the presence of Lawsoniaintracellularis. Journal of Veterinary Diagnostic Investi-gation 2003; 15 : 268–273.14. Hampson DJ. Colonic spirochaetal infections of medi-cal importance. In Proceedings of the Second Inter-national Conference on Colonic Spirochaetal Infectionsin Animals and Humans. Eddlestone, Scotland, UK,2003, p. 5.15. Munshi MA, et al. Prevalence and risk factors for Bra-chyspira aalborgi colonisation in Bali. In Proceedings ofthe Second International Conference on Colonic Spiro-chaetal Infections in Animals and Humans. Eddlestone,Scotland, UK, 2003, p. 8.16. Duhamel GE. Comparative pathology and pathogenesisof naturally acquired and experimentally induced co-lonic spirochetosis. Animal Health Research Reviews2001; 2 : 3–17.17. Trott DJ, McLaren AJ, Hampson DJ. Pathogenicity ofhuman and porcine intestinal spirochetes in on-day-oldspeciﬁc-pathogen-free chicks: an animal model of in-testinal spirochetosis. Infection and Immunity 1995; 63 :3705–3710.18. Trott DJ, et al. Population genetic analysis of Serpulinapilosicoli and its molecular epidemiology in villagesin the eastern Highlands of Papua New Guinea.International Journal of Systematic Bacteriology 1998;48 : 659–668.19. Gebhart CJ. Lawsonia intracellularis : overview and re-search update. In Proceedings of the 1st Novartis AnimalHealthcare European Swine Ileitis/Colitis Workshop.Alpbach, Austria, 2004, pp. 1–5.The occurrence of Brachyspira species and L. intracellularis in children 1045https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268806007606Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 20:02:44, subject to the Cambridge Core terms of use, available at

Survey on the occurrence of Brachyspira species and Lawsonia intracellularis in children living on pig farms

Survey on the occurrence of Brachyspira species and Lawsonia intracellularis in children living on pig farms

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